A gene regulating the flavor of morel mushrooms and screening method thereof

Abstract

A gene regulating the flavor of morels and a screening method thereof, wherein the nucleotide sequence of the gene regulating the flavor of morels is shown in SEQ ID No. 1. The invention screens the flavor-related genes of morels by different culture temperatures, and can be used to develop morel flavor enhancers to enhance the flavor of morels.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and edible fungi, and relates to a gene for regulating the flavor of morels and a screening method thereof. Background Art

[0002] Morchella spp. is a rare edible and medicinal mushroom. It is crispy and delicious, with a unique flavor. It is rich in protein, vitamins, polysaccharides and many bioactive ingredients. Morchella has been cultivated artificially for more than a hundred years, but it still faces problems such as difficult to ensure quality and unstable yield. In recent years, my country has gradually formed a field ecological cultivation model for morels and began to standardize the cultivation of morels. The environmental temperature cannot be accurately adjusted during field cultivation, and temperature stress has a significant impact on the growth of morels.

[0003] Morchella is a low-temperature mushroom, and temperature is one of the key factors for its growth and development. During the growth process, Morchella requires lower temperatures and greater temperature differences, which are convenient for stimulating mycelial differentiation. Studies have shown that low temperature stress can cause oxidative damage to edible fungi. During low temperature stress, superoxide dismutase (SOD) activity increases in the early stage and decreases in the later stage, catalase (CAT) and ascorbate peroxidase (APX) activities generally increase, and glutathione (GSH) content increases; heat stress will prompt plants to produce antioxidants, accumulate and regulate the content of compatible solutes, and induce mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) cascades. These regulations are achieved through complex interactions between members of different gene families, such as participating in cell signal transduction pathways. In existing studies, transcriptome methods are usually used to study the effects of temperature and other factors on the growth of edible fungi.

[0004] Transcriptome refers to the complete transcripts and transcript numbers formed at a certain developmental stage or physiological condition in a cell, mainly composed of ribosomal RNA (80%-90%, rRNA), transfer RNA (5%-15%, tRNA), messenger RNA (2%-4%, mRNA) and non-coding RNA (1%, ncRNA) within or between genes. Omics technology has high sensitivity and a wide range of applications, and is favored by researchers. The application of omics technology in edible fungi research is a major breakthrough in edible fungi research, which has shifted edible fungi research from artificial cultivation to basic research. In different tissue types of edible fungi, these transcriptome components have their own unique biological functions. Transcriptomic studies on different types of edible fungi can help people better reveal the mode of action and regulatory process of functional genes, thereby improving edible fungi breeding selection and cultivation practices. Summary of the invention

[0005] The purpose of the present invention is to provide a gene for regulating the flavor of Morchella and a screening method thereof.

[0006] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a gene for regulating the flavor of Morchella edodes, wherein the nucleotide sequence of the gene for regulating the flavor of Morchella edodes is shown in SEQ ID No.1.

[0007] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a method for screening genes regulating the flavor of Morchella, comprising the following steps:

[0008] Step 1: Take activated Morchella edulis blocks of uniform diameter, transfer them to the center of the culture medium, and culture them at different temperatures for the same time;

[0009] Step 2: Mark the diameter of the colony according to the extent to which the hyphae extend toward the plate and calculate the hyphae growth rate;

[0010] Step 3: Use an inverted microscope to observe the microscopic morphology of mycelium;

[0011] Step 4: Collect Morchella mycelium at different temperatures, extract RNA for transcriptome sequencing;

[0012] Step 5: Compare and analyze the sequencing sequence with the specified Morchella reference genome, calculate the gene expression level and annotation information, and obtain the differentially expressed genes of mycelium at different culture temperatures;

[0013] Step 6: Use weighted gene co-expression network analysis to construct a hierarchical clustering tree of differentially expressed genes and explore the association between different gene modules in the clustering tree and mycelium culture temperature;

[0014] Step 7: Construct a collinear network diagram based on the relationships between genes in each module and mine morel flavor-related genes involved in temperature regulation.

[0015] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0016] As a precious edible mushroom resource, Morchella is crispy and delicious, delicious, unique in flavor, nutritious, rich in protein, vitamins, polysaccharides and many bioactive ingredients, and is increasingly favored by consumers. Flavor is determined by the balance and mutual influence of various free amino acids with different flavors. Amino acids are one of the characteristic flavor compounds of Morchella, and Morchella mycelium is rich in amino acids. Morchella is a low-temperature mushroom, and temperature is one of the key factors for its growth and development. At present, there are few reports on the regulation of Morchella flavor by culture temperature. The present invention screens Morchella flavor-related genes by different culture temperatures, and can be used to develop Morchella flavor enhancers to play the role of Morchella in enhancing the flavor. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Comparison of mycelial growth rates of the tested strains at different temperatures.

[0018] Figure 2 The microscopic hyphae morphology of the tested strains at different temperatures. From top to bottom, they are H1, H5, H6, H8, H18, M4, M15, M24, MZ-14, MZ-20, MZ-22, T6, T7; from left to right, they are 5℃, 10℃, 15℃, 20℃, 25℃, 30℃.

[0019] Figure 3 Colinear network diagram of genes negatively regulated by culture temperature on Morchella mycelium. DETAILED DESCRIPTION

[0020] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0021] See also Figure 1-3 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0022] Example 1: A gene regulating the flavor of Morchella and a screening method thereof

[0023] 1. Screening the optimal temperature range for the growth of Morchella mycelium

[0024] (1) A total of 13 strains of Morchella edulis from three species, namely, M. sextelata, M. septimelata and M. importuna, were selected and activated by transferring each strain from a test tube to PDA medium using a sterile inoculation needle on an ultra-clean workbench.

[0025] (2) Take a 5 mm × 5 mm activated Morchella edulis block, place it in the center of a 90 mm diameter PDA culture medium, and culture it in a dark constant temperature incubator at 5°C, 10°C, 15°C, 20°C, 25°C, and 30°C, respectively.

[0026] (3) The extent to which mycelium extended toward the culture medium after 24 h and 48 h of growth at each culture temperature was recorded and used to calculate the mycelium growth rate in each treatment group, i.e., mycelium growth rate = (diameter measured at 48 h - diameter measured at 24 h) / d. Each treatment was repeated 5 times.

[0027] (4) The culture plates of the six temperature treatment groups were placed under an inverted microscope (Zeiss LSM 880) and the mycelial bifurcation was observed at 40× magnification. Each treatment group had five replicates.

[0028] (5) SPSS (version 26.0) software was used to analyze the data for significant differences. The optimal culture temperature for mycelial growth of the 13 tested Morchella strains was determined to be 15-20°C based on the growth rate and mycelial bifurcation.

[0029] 2. Screening of temperature-regulated flavor genes in Morchella mycelium

[0030] (1) The test strain H6 (M. sextelata) was used as a representative Morchella strain and cultured at 5°C, 10°C, 15°C, 20°C, 25°C, and 30°C for 5 days;

[0031] (2) Use a sterile scraper to collect the mycelium from the culture medium of each temperature treatment group and place it in a sterile bag. Place it in an oven to dry. Repeat 3 times for each treatment group.

[0032] (3) The dried mycelium was placed in liquid nitrogen and fully ground into powder. 100 mg of the powder was placed in a 1.5 mL centrifuge tube without RNase. 1 mL of Trizol was added, and the tube was shaken vigorously for 30 seconds and then placed on ice for 3 minutes to allow the mycelium cells to be fully lysed.

[0033] (4) Add 200 μL of chloroform, cap the tube tightly, shake vigorously for 15 seconds, let it stand on ice for 5 minutes, and centrifuge it at 12,000 rpm at 4°C for 15 minutes to separate the phases.

[0034] (5) Transfer 500 μL of the upper aqueous phase to a new centrifuge tube, add 500 μL of isopropanol, mix thoroughly, let stand on ice for 10 min, and centrifuge at 10,000 rpm at 4°C for 10 min to precipitate RNA.

[0035] (6) Add 1 mL of 75% ethanol to rinse three times, place in a centrifuge at 7000 rpm, centrifuge at 4°C for 5 min to wash the RNA and remove the supernatant.

[0036] (7) The RNA precipitate was dried at room temperature in a clean bench, and then 50 μL of DEPC water was added to fully dissolve it to obtain mycelial RNA from each treatment group.

[0037] (8) The concentration and purity of the extracted RNA were tested by Nanodrop 2000, the integrity of the RNA was tested by agarose gel electrophoresis, the RIN value was determined by Agilent 5300, and the RNA was stored at -80°C.

[0038] (9) Use Oligo dT-labeled magnetic beads to isolate mRNA from the total RNA of each treatment group. Add fragmentation buffer to randomly break the mRNA into small fragments of about 300 bp.

[0039] (10) Using mRNA as a template, reverse transcriptase was used to synthesize cDNA. End Repair Mix was then added to fill the sticky ends of the double-stranded cDNA into blunt ends, and an A base was added to the 3' end to connect a Y-shaped adapter. The product after connecting the adapter was enriched using the IlluminaNovaSeq Reagent Kit (Illumina, San Diego, CA, USA), and the target band was recovered using 2% agarose for quantification by Qubit 4.0 to obtain the final library, which was then sequenced on the IlluminaNovaSeq6000 platform.

[0040] (11) Trimmomatic (version 0.36) software was used to perform data quality control on the original sequence, including removing reads without inserts or with an N-content exceeding 10%, and removing sequences with a quality value less than 10 or a length less than 20 bp, thereby obtaining optimized high-quality clean data.

[0041] (12) The clean data after quality control were compared with the reference genome of Morchella (GCF_020137385.1_ASM2013738v1) to analyze the gene expression level and functional annotation information.

[0042] (13) The mycelium cultured at 20°C was used as the control group to obtain the differentially expressed genes of the mycelium at different culture temperatures.

[0043] (14) Weighted gene co-expression network analysis (WGCNA) was used to construct a hierarchical clustering tree of differentially expressed genes. The co-expressed gene modules were found according to the different branches of the clustering tree, and the correlation between each gene module and the mycelial culture temperature was explored.

[0044] (15) Based on the relationships between genes in each module, a collinear network diagram was constructed to display the regulatory relationship of genes within the module and to explore the flavor-related genes of morels involved in temperature regulation.

[0045] 3. Results Analysis

[0046] (1) Test strains

[0047] Table 1 Sources of tested strains

[0048]

[0049] (2) Effects of different temperature treatments on mycelial growth rate

[0050] Table 2 Mycelial growth rate of the tested strains at different temperatures

[0051]

[0052] Note: Data are expressed as mean ± variance (n = 10); different lowercase letters indicate significant differences between the same strain at different temperatures (P < 0.5)

[0053] Figure 1 This is a bar chart of the growth rates of different Morchella strains at the same temperature. It can be seen from the figure that the growth rates of various varieties are significantly different below 20°C, while when the temperature is above 20°C, the growth rate differences of various varieties become smaller. At 20°C, the growth rate of 9 strains reached 14.00 mm / d.

[0054] (3) Effects of different temperature treatments on the microscopic morphology of Morchella mycelium

[0055] Overall, the number of mycelium tip bifurcations increased with increasing temperature ( Figure 2). When the temperature is low, the hyphae are loose and straight, but as the temperature rises, the hyphae bend and kink, and the hyphae density increases. Taking H8 as an example, at 5℃, the hyphae are straight, the front branches are short, and the number of forks is small; at 10℃, the front branches of the hyphae are extended, and the number of forks is small; the same is true at 15℃. It is speculated that when the temperature is low, the hyphae concentrate on growth and reduce branches in order to maintain vitality. At 20℃, the number of hyphae forks increases, and the hyphae density increases; at 25℃, the number of hyphae forks is the largest, and the hyphae is the densest. At this time, the temperature is high, the hyphae forks quickly, and the hyphae's adaptability to the environment is increased. At 30℃, the hyphae bending phenomenon is obvious.

[0056] (4) Differentially expressed genes in mycelium in different temperature treatment groups

[0057] Taking the 20℃ group as the control group, there were 2334 genes in the 10℃ group, of which 669 were up-regulated and 1665 were time-regulated; there were 1950 genes in the 15℃ group, of which 752 were up-regulated and 1198 were time-regulated; there were 1655 genes in the 25℃ group, of which 1129 were up-regulated and 526 were time-regulated; there were 1861 genes in the 30℃ group, of which 1299 were up-regulated and 562 were time-regulated.

[0058] Table 3 Differentially expressed genes in different temperature treatment groups

[0059]

[0060] (5) WGCNA analysis

[0061] A total of 739 genes with significant negative correlation with culture temperature were obtained through WGCNA (r = -0.896, p < 0.001). A collinear network diagram was constructed for these negatively regulated genes, as shown in Figure 3 As shown. The collinear network diagram consists of 122 nodes and 4,710 lines, and all nodes are divided into three main modules, namely module I, module II and module III. The three modules contain 70, 38 and 14 genes, respectively (Table 4). Through gene function annotation, the gene H6S33_001094 (flavo protein WrbA) related to the flavor of Morchella was found in module III, and the nucleotide sequence of the sequence of gene H6S33_001094 is shown in SEQ ID No.1. This result shows that the flavor of Morchella decreases with the increase of mycelium culture temperature.

[0062] Table 4 Detailed information on gene functions in each module

[0063]

[0064]

[0065]

[0066]

[0067] 4. Morchella, as a precious edible mushroom resource, is crispy and delicious, delicious, unique in flavor, nutritious, rich in protein, vitamins, etc., polysaccharides and many bioactive ingredients, and is increasingly favored by consumers. Flavor is determined by the balance and mutual influence of various free amino acids with different flavors. Amino acid is one of the characteristic flavor compounds of Morchella, and Morchella mycelium is rich in amino acids. Morchella is a low-temperature mushroom, and temperature is one of the key factors for its growth and development. At present, there are few reports on the regulation of Morchella flavor by culture temperature. This embodiment screens Morchella flavor-related genes by different culture temperatures, which can be used to develop Morchella flavor enhancers to play the role of Morchella in enhancing flavor.

[0068] SEQ ID No.1:

[0069] .

[0070] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A gene for regulating the flavor of Morchella, characterized in that: The nucleotide sequence of the gene regulating the flavor of Morchella is shown in SEQ ID No.

1.

2. A method for screening genes that regulate the flavor of Morchella, characterized in that: The following steps are involved: Step 1: Take activated Morchella edulis blocks of uniform diameter, transfer them to the center of the culture medium, and culture them at different temperatures for the same time; Step 2: Mark the diameter of the colony according to the extent to which the hyphae extend toward the plate and calculate the hyphae growth rate; Step 3: Use an inverted microscope to observe the microscopic morphology of mycelium; Step 4: Collect Morchella mycelium at different temperatures, extract RNA for transcriptome sequencing; Step 5: Compare and analyze the sequencing sequence with the specified Morchella reference genome, calculate the gene expression level and annotation information, and obtain the differentially expressed genes of mycelium at different culture temperatures; Step 6: Use weighted gene co-expression network analysis to construct a hierarchical clustering tree of differentially expressed genes and explore the association between different gene modules in the clustering tree and mycelium culture temperature; Step 7: Construct a collinear network diagram based on the relationship between genes in each module, and mine morel flavor-related genes involved in temperature regulation; A gene for regulating the flavor of Morchella edodes was obtained, and the nucleotide sequence was shown as SEQ ID No.1.

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